1. Field of the Invention
The invention concerns a design principle for a minimalistic expression construct which contains no genetic information other than the information to be expressed apart from promotor and terminator sequences which are necessary for the control of expression Such minimal expression constructs are to be used for molecular-medical applications, specifically genetic vaccination, tumor therapy, and -prophylaxis.
The design principle is to be used for the construction of expression constructs for the expression of MHC-I or MHC-II presentable peptides, cytokines, or components of the regulation of the cell cycle, or for the synthesis of regulative RNA molecules and antisense RNA, ribozyme or mRNA-editing-RNA. Furthermore, an important aspect of the invention is that the construction principle allows for the covalent linking of the expression construct, e.g. with peptides, proteins, carbohydrates or glycopeptide ligands, as well as particles which allow for the transfer of the constructs into cells by ballistic transfer especially into dermis, muscle tissue, pancreas, and the liver.
2. Background Information
The invention is to be used especially in two related fields: somatic gene therapy and genetic vaccination. These two meet in the field of immuno gene therapy of oncological conditions. Whereas classical gene therapy intends to substitute missing or defective genes, immuno gene therapy intends to activate the immune system of the patient against tumor specific antigens. In malignant melanoma and some other tumors, a number of tumorspecific antigens have been identified which can be recognized by cytotoxic T-lymphocytes (Van den Eynde B. and Brichard V. G., Current Opinion in Immunology (1995) 7: 674–681). In most cases these are fragments of mutated proteins, which are either relevant for tumor development and -promotion, or are fragments of proteins from a changed metabolism of the tumor cell (Stüber et al., Eur. J. Immunol. (1994) 24: 765–768). In the case of melanoma, the presented peptides often derive from proteins from the melanocyte-specific differentiation. Approaches which make use of the activation of the immune system against such tumor specific antigens are in need of methods which enable the antigenic epitopes to be overexpressed in non-tumor cells, such as antigen-presenting cells (macrophages, dendritic cells) Alternatively, genes which control the expression of peptide-presenting proteins, such as CIITA or ICSBP are of great importance.
Laboratory experiments and clinical studies, in which such peptides have been used for the induction or amplification of a tumor specific cytotoxic response, concentrate on conventional vaccination protocols, in which the corresponding peptides are being used (Strominger J., Nature Medicine, (1995) 1:1.179–1.183). Alternatively, antigen-presenting cells such as dendritic cells, were incubated with high concentrations of such peptides. Thereby, the peptides originally present on the MHC-complex were exchanged for tumor specific peptides (Grabbe et al., Immunology Today (1995) 16:117–121).
The term genetic vaccination (immunization) describes the utilization of an experimental finding which first was debated as a scientific artefact, but has recently been corroborated in a number of biomedical problems (Piatak et al., Science 259 (1993): 1745–1749). If an expression plasmid for mammalian cells is injected into skin or muscle, there is, albeit in very low efficiency, an expression of the corresponding gene close to the injection site. If the expression product is a protein alien to the organism (xenogenic or allogenic protein), uptake and presentation of fragments of the expressed protein (oligopeptide) by antigen-presenting cells (APC) takes place, probably by way of local inflammation, Depending on local cytokine patterns and the type of cells in which the plasmid is expressed (presentation by MHC-I or MHC-II), there is an induction of an immune reaction along the TH1 or TH2 pathway (Wang et al., Human Gene Therapy 6 (1995): 407–418), which eventually leads to the proliferation of cytotoxic T-cells or to the production of soluble antibodies. The transfection of dendritic cells with expression constructs for antigenic peptides ex-vivo is included in the term genetic vaccination in this context (Schadendorf et al. Molecular Medicine Today, 2 (1996): 144–145).
Such genetic vaccination avoids the numerous risks of conventional immunization approaches. Many approaches are known in gene therapy that are designed to effect therapeutic or prophylactic effects by the transfer of genetic information into cells. These approaches have not only been demonstrated in animal experiments, but also in numerous clinical studies in patients, an example being the so-called ballisto magnetic vector system (EP0686697 A2) for the transfection of conventional, plasmid-based expression constructs. The ballisto magnetic vector system was employed by the inventors of this application in three clinical phase I/II studies for the production of interleukin-7 (IL-7), interleukin-12 (IL-12) or granulocyte-macrophage-colony stimulating factor (GM-CSF) expressing tumor cells. In the case of expression of IL-12, separate expression constructs for the genes of the two IL-12 subunits were transferred ballisto-magnetically at the same time.
With the maturing of this new discipline, however, the methodological repertoire for gene therapy demands critical inspection. A fundamental aspect of this question is the sequence information contained in conventionally employed DNA constructs . If such expression constructs are to be employed in a great number of patients, and possibly more than once, safety aspects, especially those related to immunological concerns, will come to bear heavily. The conventionally used expression constructs are derivatives of eucaryotic expression plasmids. These have two fundamental disadvantages: their size, which inhibits fast transport into the cell's nucleus, and the presence of sequences which are not needed for the intended use. Expression constructs used so far contain constitutively expressed genes, i.e. for resistance against cytostatica which serve as selection markers, and in some cases sequences for the episomal replication in the target cell. The expression of these genes leads to an unwanted background of transfected genetic information. Furthermore, apart from the promotor-gene-terminator structure which is to be expressed, these constructs carry at least the sequences needed for bacterial replication, since the plasmids are propagated in bacteria. These sequences are not needed for the intended use, either.
It is obvious that conventional expression constructs lead not only to the expression of the desired gene, but also to the biosynthesis of xenogenic proteins, even if their prokaryotic promoters show very low activity in mammalian cells. With longer or repeated application it can be assumed that the desired immune response is masked by such contaminating gene products, and significant immunological complications can occur.
Another problem in the application of gene therapeutic methods concerns the method by which the genetic material to be transferred is brought into the cell. For reasons of efficiency, immunological safety, and broad applicability across a wide spectrum of cell types, the method of ballistic transfer is preferred. A fundamental advantage of ballistic transfer, compared to alternative transfection methods, is that the method is applicable across a broad spectrum of different cells or tissues. Another disadvantage of methods currently used for the transfection of eukaryotic cells, such as electroporation or lipofaction, is that the treatment brings the substance to be transported only across the plasma membrane, the first barrier, which shields the cell from its environment. However, for most substances interacting with the regulative function of the cell, it is important to get from the cytoplasma across the nuclear membrane into the nucleus. This membrane is biophysically fundamentally different from the plasma membrane, and methods such as electroporation or lipofection do not lead to a passage through this membrane. The reason why these methods nonetheless lead to expression of recombinant nucleic acid constructs transfected into the cells in a part of the cell population, is the fact that in the act of cell division, the nuclear membrane is rendered permeable In consequence, methods such as electroporation or lipofaction only lead to transfection of cells which divide. Therefore these methods are not applicable to the transfection of many slowly or non-dividing cells, which can be interesting in the context of gene therapy, such as stem cells of the immune system or the heamatopoietic system, muscle cells, cells of exocrine or endocrine organs and their accompanying cells. The also commonly used and very efficient transfection method of retroviral transport of genetic material suffers the great disadvantage of targeting the tranfected cells for a possible cytotoxic reaction by the host organism, which probably limits the applicability of this method for gene therapeutic approaches.
The method of ballistic transfer has been used for the ex-vivo treatment of autologous and allogenic patient cells (Mahvi et.al.; Human Gene Therapy 7 (1996) 1535–43). However, when treating cells in tissue, a method which should be advantageous especially for the oncotherapeutic treatment of solid tumors or the mass prophylaxis against infections by genetic vaccination, the state of the art has disadvantages. The method of ballistic transfer makes use of DNA adsorbed to microprojectiles. When transfecting skin or other tissues, the penetration depth of the DNA constructs is lower than the penetration depth of the projectiles. DNA is desorbed soon after impact on the tissue. Only the uppermost tissue layer in the direction of the projectiles is transfected, although the projectiles themselves enter much deeper into the tissue. When transfecting solid tumor tissue (colon carcinoma, rectum carcinoma, reno-cell carcinoma and others), it has been found that, with suitable adaption of the parameters, the microprojectiles enter up to five cell layers deep into tissue slices. The transfected cells, however, (visible as fluorescent cells when transfected with a recombinant expression construct containing a green fluorescent protein from aequrea sectoria) were all found in the uppermost cell layer facing the impact of the microprojectiles. A more stable coupling of the DNA constructs to the surface of the microprojectiles would be desirable in order to avoid the desorption of the substance to be transported. In this way only, the application of gene therapeutic approaches to solid tumors would be realistic, since only the transfection of tumor slices in the depth of the tissue enables a sufficient number of treated cells to be achieved. It is also imaginable that a combination of different coupling protocols enables the release of different genetic information within the same cell population at different timepoints. For these and numerous other applications, microparticles which bring the substance to be transported all the way into the hit cell and then make the substance available to the cell, would be very desirable.
U.S. Pat. No. 5,584,807 (McCabe) describes an instrument in form of a gas pressure operated gun for the introduction of genetic material into biological tissue, in which gold particles are used as carrier material for the genetic information, without making reference to the nature of the genetic material in particular. U.S. Pat. No. 5,580,859 and U.S. Pat. No. 5,589,466 (Felgner) describe a method for the introduction of DNA into mammalian cells in the context of gene therapy. Naked DNA sequences coding for physiologically active proteins, peptides or polypeptides and are under the control of a promotor are injected directly into cells. Naked DNA refers to sequences that are free of other genetic material like viral sequences. DNA is expressed in these cells and serves as vaccine.
WO 96/26270 (Rhône-Poulenc Rorer S. A.) describes a circular double-stranded (supercoiled) DNA molecule, containing an expression cassette coding for a gene and controlled by a promoter and a terminator. This system is employed in vaccination in the context of gene therapy, also.
EP 0 686 697 A2 (Soft Gene) concerns a method for the enrichment of cells modified by ballistic transfer, and describes the technological background, the related problems, and the solutions found so far. The basic method of ballistic transfer is described herein. A device useful for the execution of this method is described in EP 0 732 395 A1.
The ballistic particles are gold particles with a diameter of either 1 μm or 1,5 μm (EP 0 686 697 A2), chosen depending upon the cell type. These gold particles are coated with superparamagnetic particles of roughly 30 nm diameter. The superparamagnetic particles at the same time furnish a useful surface for the coating with biomolecules. The use of magnetic particles enables subseqent separation.
Furthermore, dumbbell-shaped nucleic acid constructs are known that are characterized by the following features: They are short (10–50 bp double-stranded DNA) nucleic acid constructs, which were made for structure research or as double-stranded oligomers with improved nuclease resitence used for scavenging of sequence specific DNA ligands (Clusel et. al.; Nucleic Acids Res. 21 (1993): 3405–11; Lim et. al., Nucleic Acids Res. 25 (1997): 575–81).
Longer DNA molecules, which can exist throughout parts of their replication cycles as dumbbells, are known in nature as mitochondrial genomes of some species, such as ciliatae and yeasts (Dinuel et. al., Molecular and Cell Biology, 13 (1993): 2315–23). These molecules are about 50 kb in size and have a very complex genetical structure. Likewise, a closed covalent linear structure is known from vaccinia virus.
Peptide-nucleic acid-linkages with localization sequences are known for short DNA oligomers. Morris et al. (Nucleic Acids Res. 25 (1997): 2730–36) describe the coupling of oligomers 18–36 base pairs in length, with a 27 amino acid residues containing peptide, which contains the nuclear localization sequence from SV40 as well as a signal peptide from HIV-gp41 responsible for the fusion with CD4-positive cells.
The use of peptide chains for crossing the endosomal membrane has been investigated by several groups. The 23 N-terminal amino acids of haemagglutinine were adsorbed by non-covalent interactions to expression plasmids in order to facilitate the uptake of these complexes into the cytosol after endosomal uptake (Plank et.al., J.Biol.Chem. 269, 12918, (1994)). The covalent attachment of antisense desoxyoligonucleotides to haemagglutinine peptide is described by Bongartz et al. (Nuc.Acids Res. 22, 4681, 1994).